1. Field of the Invention
This invention relates to spectroscopic measurements and, in particular, spectroscopic measurements involving emission.
2. Art Background
Spectroscopic measurements are utilized in a host of analytical techniques. For example, such measurements are often utilized to detect the presence of various spectroscopically active biological species. Spectroscopic measurements are also utilized, in another common application, to detect the presence of various sample components exiting a chromatographic column. Generally, in analytical spectroscopic techniques, irrespective of the particular application, the presence of a specific chemical species is detected through the interaction of electromagnetic radiation with the species. The most commonly employed spectroscopic methods are absorption techniques--monitoring of absorbed electromagnetic radiation--and fluorescent techniques--the monitoring of emitted radiation including but not limited to fluorescence and phosphorescence. Although absorption techniques are quite useful, generally fluorescence measurements are significantly more sensitive. Thus, the utilization of fluorescence techniques potentially allows the detection of a greater variety of chemical entities at lower concentrations.
In fluorescence techniques the entity to be detected is excited utilizing incident electromagnetic radiation. Upon relaxation of this excitation, light is emitted that is indicative of the species being monitored. Although fluorescence measurements offer the possibility of greater sensitivity, quenching of emission from a potentially fluorescing species by interaction with a second entity often significantly diminishes or eliminates this advantage. Although some quenching is induced by interaction of a solid sample with its gaseous environment, i.e., air, this interaction resulting, for example, from adsorption of gas, e.g., oxygen, on the solid sample surface is easily eliminated by operating in a non-quenching inert atmosphere, e.g., making the measurement under nitrogen or helium. The most significant quenching processes, however, involve the interaction of a quenching entity with the excited species in a condensed phase. (A condensed phase for purposes of this disclosure is a solid or a phase whose density is no less than 50 percent of the density measured for the same composition in the solid phase. Interaction refers to effects produced by the presence of one entity in the vicinity of another where these two entities are not the specific combination of a solid and a gas absorbed directly from a gaseous environment onto the surface of the solid.) The quenching in these significant systems occurs through various interactions--in solids, for example, by contact with a quencher from a source other than by direct adsorption from a gaseous environment and in other condensed phases by, for example, contact with the quencher, e.g., through dissolution in a solution containing the fluorescing species. In one specific example, although rhodamine B fluoresces intensely upon excitation with blue-green light, the presence of iron quenches this emission by conversion to a non-radiative state. This undesirable quenching is often caused by reagents typically utilized in analytical techniques or in the case of biological investigations by chemical species typically present in biological systems. Thus, for many significant applications, although sensitive measurements are quite desirable, the additional sensitivity provided by fluorescence measurements is not available and absorption techniques are being employed.